Abstract

The voltage-gated sodium channel Na_v1.7 plays a crucial role in pain, and drugs that inhibit hNa_v1.7 may have tremendous therapeutic potential. ProTx-II and huwentoxin-IV (HWTX-IV), cystine knot peptides from tarantula venoms, preferentially block hNa_v1.7. Understanding the interactions of these toxins with sodium channels could aid the development of novel pain therapeutics. Whereas both ProTx-II and HWTX-IV have been proposed to preferentially block hNa_v1.7 activation by trapping the domain II voltage-sensor in the resting configuration, we show that specific residues in the voltage-sensor paddle of domain II play substantially different roles in determining the affinities of these toxins to hNa_v1.7. The mutation E818C increases ProTx-II's and HWTX-IV's IC₅₀ for block of hNa_v1.7 currents by 4- and 400-fold, respectively. In contrast, the mutation F813G decreases ProTx-II affinity by 9-fold but has no effect on HWTX-IV affinity. It is noteworthy that we also show that ProTx-II, but not HWTX-IV, preferentially interacts with hNa_v1.7 to impede fast inactivation by trapping the domain IV voltage-sensor in the resting configuration. Mutations E1589Q and T1590K in domain IV each decreased ProTx-II's IC₅₀ for impairment of fast inactivation by ∼6-fold. In contrast mutations D1586A and F1592A in domain-IV increased ProTx-II's IC₅₀ for impairment of fast inactivation by ∼4-fold. Our results show that whereas ProTx-II and HWTX-IV binding determinants on domain-II may overlap, domain II plays a much more crucial role for HWTX-IV, and contrary to what has been proposed to be a guiding principle of sodium channel pharmacology, molecules do not have to exclusively target the domain IV voltage-sensor to influence sodium channel inactivation.